Wang Yilun, Cao Zheng, Xu Wei, Kemp Marlo D, McAdory Brenda S, Newkirk Robert F, Ivy Michael T, Townsel James G
Department of Biological Sciences, Tennessee State University, Nashville, TN 37209, USA.
Gene. 2004 Feb 4;326:189-99. doi: 10.1016/j.gene.2003.11.005.
We describe herein the cloning of a group of syntaxins in Limulus that are associated with the plasma membrane. Initially, multiple degenerate oligonucleotide primers (DOP) and probes were designed from sequences of known plasma membrane associated syntaxins. Combined experiments using reverse transcriptase-polymerase chain reaction (RT-PCR), colony hybridization and reverse dot blot yielded three distinct probes. Subsequently, two cDNA libraries derived from the Limulus central nervous system (CNS) were screened and four distinct isoforms, designated Limulus syntaxin (Lim-syn) 1A, 1B, 1C and 1D, were obtained from forty cloned full-length sequences. The predicted amino acid (aa) sequences 1-265 were identical for Lim-syn 1A, 1C and for Lim-syn 1B, 1D, respectively. A comparison of the 265 aa cytoplasmic segments for the two subgroups Lim-syn 1A/1C and Lim-syn 1B/1D differed at 13 aa residues within this sequence. Lim-syn 1A and 1B contained 290 aa residues, and both contained a transmembrane domain (TMD, 267-288) and a myristylation-like site (286-290) at the C-termini. Lim-syn 1C (291 residues) contained only the TMD whereas Lim-syn 1D was truncated (277 residues) and had neither a TMD nor a myristylation-like site. All Lim-syn isoforms showed great identity with syntaxin 1-homologs (syntaxin 1A/1B) from various other species. Ribonuclease protection assay (RPA) analyses revealed distinctive expression patterns for individual Lim-syn transcripts but all were detectable in the CNS. Moreover, the antibody (anti-Lim-syn-1) produced against aa 133-145 epitope of Lim-syn identified a protein of approximately 35 kDa found only in CNS tissues.
我们在此描述了在鲎中克隆的一组与质膜相关的 syntaxin。最初，根据已知的与质膜相关的 syntaxin 序列设计了多个简并寡核苷酸引物（DOP）和探针。使用逆转录聚合酶链反应（RT-PCR）、菌落杂交和反向斑点杂交的联合实验产生了三种不同的探针。随后，筛选了两个来自鲎中枢神经系统（CNS）的 cDNA 文库，从 40 个克隆的全长序列中获得了四种不同的同工型，命名为鲎 syntaxin（Lim-syn）1A、1B、1C 和 1D。Lim-syn 1A、1C 的预测氨基酸（aa）序列 1-265 分别相同，Lim-syn 1B、1D 的预测氨基酸序列 1-265 也分别相同。Lim-syn 1A/1C 和 Lim-syn 1B/1D 这两个亚组的 265 个 aa 细胞质区段在该序列中的 13 个 aa 残基处存在差异。Lim-syn 1A 和 1B 包含 290 个 aa 残基，并且在 C 末端均含有一个跨膜结构域（TMD，267-288）和一个类豆蔻酰化位点（286-290）。Lim-syn 1C（291 个残基）仅含有 TMD，而 Lim-syn 1D 被截短（277 个残基），既没有 TMD 也没有类豆蔻酰化位点。所有 Lim-syn 同工型与来自其他各种物种的 syntaxin 1 同源物（syntaxin 1A/1B）具有高度同源性。核糖核酸酶保护分析（RPA）显示各个 Lim-syn 转录本具有独特的表达模式，但在 CNS 中均可检测到。此外，针对 Lim-syn 的 aa 133-145 表位产生的抗体（抗 Lim-syn-1）鉴定出一种仅在 CNS 组织中发现的约 35 kDa 的蛋白质。
